Short bowel syndrome (SBS) due to surgical resection for Crohn's disease, ischemia, trauma, necrotizing enterocolitis or other disorders is a major cause of morbidity, mortality and high health care costs in the U.S. Short bowel patients are frequently dependent on parenteral nutrition to meet some or all of their nutritional requirements. Following intestinal resection, the remaining small bowel epithelium mounts an adaptive response that increases villus height, crypt depth and enhances nutrient and electrolyte absorption. The ultimate goal of the proposed studies is to identify interventions that can enhance intestinal adaptation and thus reduce or eliminate dependence on parenteral nutrition. Little is known about specific mediators of the stem cell/proliferative and the functional adaptive response to intestinal resection. Accordingly, a critical unmet need is to understand the signaling pathways (nutritional and microbial) that modulate intestinal stem cell interactions with the local and luminal environments following small bowel resection. We will use cohorts of both pediatric and adult SBS patients to test the overarching hypothesis that SBS patients who successfully wean from TPN manifest shifts in microbial communities associated with altered serum and fecal BA metabolomic profiles and signaling, as well as BA synthesis rates (C4 determination) that predict functional intestinal adaptation. Aim 1 will test the hypothesis that distinctive microbiome and BA metabolic profiles and compensated BA synthesis are associated with successful intestinal adaptation and permit weaning and independence from (PN). Aim 2 will test the hypothesis that changes in BA signaling pathways associated with altered gut microbiome, identified in Aim 1, promote gut stem and crypt cell proliferation (CCP), as well as functional and metabolic adaptation. Because adaptation in adults continues for up to 2 years following resection, we will examine the temporal evolution of these metabolic and environmental signaling factors in SBS patients. Accordingly, our overarching objective is to identify key BA metabolomic and metagenomic changes that predict successful adaptation and to use informative preclinical models to identify the mechanisms of BA signaling and stem/CCP and functional adaptation. This objective will be accomplished through two aims. Aim 1. Identify metagenomic and BA metabolomic (blood and stool BA species signatures; BA synthesis rates) in pediatric and adult SBS patients for up to two years after small bowel resection, and define their functional relationships with body composition, intestinal stem/crypt cell proliferative responses, and clinical outcomes including independence from PN. Aim 2. Identify the mechanisms by which changes in the microbiome and the associated shift in BA signaling metabolites modulate morphologic and functional /metabolic adaptation using in vitro stem/enteroid cultures and gnotobiotic mice. Gnotobiotic Lgr5eGFP mice will be gavaged with fecal microbial transplants from patients who wean (adapters) from PN compared to those who cannot wean (non-adapters) and assessed for the ability to harvest energy. Normal and SBS patient stem cell cultures will be incubated with individual and combinations of bile acid metabolites as well as high affinity FXR ligands and assessed for stem cell survival, enteroid growth, budding and morphologic differentiation. Together the proposed studies are significant as they will help to identify modifiable luminal factors and bile acid metabolic pathways that may enhance adaptation and lead to innovative therapies for SBS.